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Development of Novel Chimeric Hepatitis C Virus (HCV) Cell Culture Systems Derived from Intergenotypic 1b/2a (Chinese Isolates/JFH1) and 1a/2a (H77 Strain/JFH1) Recombinant Genome

Author: LuSha
Tutor: ShenXiaoLing；BoXiaoZhen
School: Inner Mongolia Medical College
Course: Immunology
Keywords: Hepatitis C Virus Chimeric virus Cell culture
CLC: R373
Type: Master's thesis
Year: 2009
Downloads: 11
Quote: 0
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Abstract


Purpose through the development of chimeric hepatitis C virus ( HCV ) 1a and 1b subtypes envelope protein gene plasmid , the establishment of a chimeric HCV in vitro cell culture model for the screening of antiviral drugs for HCV genotype and HCV antigen iMMUNIZATION provide an effective model . Method 1 . Using fusion PCR technology , the introduction of the JFH1 plants (2a) gene skeleton the Chinese HCV Hebei isolates (1b) and H77 strain (1a) of the total length of the envelope protein gene ( E1/E2 ) by restriction enzyme digestion, restructuring and other means the building envelope protein gene region of mutual replacement chimeric viruses ( of 1b/2a and a 1A/2A ) full - length genome plasmid ; 2.HCV ( 1b/2a ) and HCV ( 1A/2A ) full - length genome plasmid by the Xbal single enzyme the tangent linear after in vitro transcription to obtain full-length viral RNA , the the RNA transfection Huh7.5.1 cells , indirect immunofluorescence , Western blot assay . 1 successfully constructed containing the chimeric virus 1b/2a 1A/2A full-length cDNA recombinant plasmid Phe- bei ( E1E2 ) / JFH1 and PH77 ( E1E2 ) / the JFH1 ; 2 . Were pHeBei (E1E2) / JFH1 pH77 (E1E2) / JFH1 as a template , in vitro transcription of virus RNA , the RNA transfection human hepatoma cell Huh7.5.1 cells , indirect immunofluorescence assay 72h after transfection positive cells was 1 % to 3 % , 5d after positive cells rate of 20 % to 50 % ; Western blot was used to detect the expression of intracellular HCV non-structural proteins NS3 and structural proteins Core . Conclusion This study is the first in the country to establish a the chimeric HCV isolates ( Hebei strains ) gene of HCV cell culture models , viral RNA replication and expression in cultured cells to produce chimeric HCV chimeric HCV in cell - to - cell spread .

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CLC: > Medicine, health > Basic Medical > Medical Microbiology ( pathogenic bacteriology,pathogenic microbiology ) > Human Virology ( pathogenic virus)
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